Download Measuring Mitochondrial Membrane Potential using

Measuring Mitochondrial Membrane Potential using
the FLUOstar Omega Microplate Reader
Heather Mortiboys and Oliver Bandmann
Academic Neurology Unit, Medical School, University Of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK.
Application Note 175
Rev. 07/2008
Mitochondrial membrane potential measured in fibroblast
cells
Tetramethylrhodamine methylester fluorescence directly
proportional to the membrane potential
Performed on BMG LABTECH’s FLUOstar Omega in 96 and
384 well plate format
Introduction
Measurement of the mitochondrial membrane potential is useful
in a wide variety of research areas and mitochondrial dysfunction is
implicated in diseases such as cancer, diabetes, Parkinson’s disease,
and stroke.
In most eukaryotic cells the majority of ATP production is
via oxidative phosphorylation by the respiratory chain. In this way,
sugars, such as glucose, and free fatty acids are oxidised, resulting
in the pumping of protons across the inner mitochondrial
membrane, creating an electro-chemical gradient (the mitochondrial membrane potential). This is in turn used by
complex V of the respiratory chain to generate ATP.1 Therefore
the mitochondrial membrane potential makes up a large part
of the bioenergetic state of the cell and it is changed directly
depending on the cells energy needs. For example fast growing
tumour cells have a much higher mitochondrial membrane
potential than WT cells, and in turn quiescent or differentiated
cells have a still lower membrane potential.2
The ability to accurately measure mitochondrial membrane
potential can give invaluable information about the general health
and function of the mitochondria, in particular of the overall
function of the respiratory chain and the potential of the
mitochondria to generate ATP and provide energy for other cellular
components.3
The number of factors which can influence this potential are
vast and include many cellular components outside the
mitochondria, for example interactions with the activity of the
proteasome. In addition, the ability of a cell to maintain its
mitochondrial membrane potential can mean the difference
between survival of the cell, entry into an apoptotic cell death
or a necrotic cell death process.4 Intact mitochondrial membrane
potential is also required for the cell to enter apoptosis.
Mitochondrial membrane potential is usually measured using
non-invasive cationic dyes. These dyes are sequestered into
the mitochondrial matrix in amounts directly proportional to the
membrane potential and can then be measured using standard
fluorescent techniques, including fluorescence microscopy, flow
cytometry or using a microplate reader for high through put assays.
BMG LABTECH’s FLUOstar Omega microplate reader was used in a
high throughput screening assay measuring mitochondrial
membrane potential in human cells. The results presented here
were gained using primary human fibroblast cells from controls.
Materials and Methods
Human fibroblast cells obtained from Coriell Cell Repositories
Black 96 or 384 well plates with transparent bottom from Greiner
(Art. No. #655090 and #781091)
Tetramethylrhodamine methyl ester (TMRM), Sigma #T5428
Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), Sigma
#C2759
Ethidium homodimer fluorescent dye, Invitrogen, #E1169
FLUOstar Omega microplate reader, BMG LABTECH, Offenburg,
Germany (Figure 1)
Fig. 1: BMG LABTECH’s multidetection plate reader FLUOstar Omega
Fibroblasts were plated at 40% confluency in 96 or 384 well plates
in minimal growth medium MEM with 10% FBS, 100 IU/mL penicillin,
100 µg/mL streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine,
0.1 mM amino acids, 50 µg/mL uridine and 1 X MEM vitamins.
24 hours later cells were changed into galactose culture medium
MEM (without glucose), supplemented with 10% FBS, 100 UI/mL
penicillin, 100 µg/mL streptomycin and 0.9 mg/mL galactose, as
described before.5
After further 24 hours of growth the mitochondrial membrane
potential was measured using the fluorescent dye Tetramethylrhodamine methyl ester (TMRM) as described before.3 Briefly the
dye was loaded onto cells at 150 nM in assay buffer (80 mM NaCl,
75 mM KCl, 25 mM D-glucose, 25 mM HEPES, pH 7.4), placed at 37°C
for 5 minutes, washed 4 times in PBS and measured on a
FLUOstar Omega microplate reader (Excitation: 544 nm and
Emission: 590 nm, bottom reading with 50 flashes per well).
Initially TMRM is loaded into the cell via the plasma membrane and
subsequently the TMRM dye is sequestered into the mitochondria
as the mitochondrial matrix is the most negatively charged part of
the cell. Therefore changes in plasma membrane potential could
influence the results obtained using TMRM. In order to control for
this, each assay is performed in parallel as above plus 10 µM
carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which collapses
the mitochondrial membrane potential. All data is expressed as the
total TMRM fluorescence minus the CCCP treated TMRM fluorescence.
It is important to quantify and control for cell number, especially
when comparing results between cell lines, therefore cell number
is measured using 1 µM ethidium homodimer fluorescent dye in a
parallel plate after freeze thawing (Excitation: 544 nm and Emission:
645 nm, bottom reading with 50 flashes per well).
Results and Discussion
Figure 2 shows the mitochondrial membrane potential in 6 control
fibroblast cell lines, each measured in triplicate on separate occasions
and showing the lack of variation between control cell lines.
Conclusion
These results show that the FLUOstar Omega is suitable for
measuring mitochondrial membrane potential in both 96 and
384 well plate formats. In addition, prolonged low dose treatment
of healthy control fibroblast with a mitochondrial toxin causes a
significant reduction in mitochondrial membrane potential.
The results are reproducible across a number of different control
fibroblast lines and the results of the rescue of this defect with the
experimental compound provide further evidence that this assay
can be used for high thoughput screening using BMG LABTECH’s
FLUOstar Omega.
References
Fig. 2: Mitochondrial Membrane Potential measured using TMRM fluorescence
in 6 different control fibroblast lines. The fluorescence values were
corrected for the cell number.
Figure 3 shows the reduction in membrane potential of the same
control fibroblasts after treatment with mitochondrial toxin ‘X’ for
72 hours. This demonstrates the range at which the assay is sensitive, creating a large ‘therapeutic window’. Also in this figure the
control cells have been treated with an experimental compound ‘Y’
which rescues the reduction in mitochondrial membrane potential
caused by treatment with the mitochondrial toxin ‘X’.
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Adam-Vizi V (2005) Production of reactive oxygen species in brain
mitochondria: contribution by electron transport chain and nonelectron transport chain sources. Antioxid. Redox Signaling 7
(9-10), 1140-1149.
Huang SG. (2002) Development of a high throughput screening
assay for mitochondrial membrane potential in living cells.
J. Biomol. Screen. 7, 383-389.
Brown GC (1999) Nitric Oxide and Mitochondrial Respiration.
Biochim. Biophys. Acta 1411 (2-3), 251-269.
Carelli V, Rugolo M, Sgarbi G, Ghelli A, Zanna C, Baracca A, Lenaz G,
Napoli E, Martinuzzi A, Solaini G (2004) Bioenergetics shapes
cellular death pathways in Leber’s hereditary optic neuropathy:
a model of mitochondrial neurodegeneration. Biochim. Biophys.
Acta 1658, 172– 179.
Hofhaus G, Johns DR, Hurko O et al. (1996) Respiration and growth
defects in transmitochondrial cell lines carrying the 11778
mutation associated with Leber’s hereditary optic neuropathy.
J. Biol. Chem. 271, 13155-13161.
Fig. 3: Comparison of mitochondrial membrane potential in 6 untreated
control fibroblast lines (norm), treated with mitochondrial toxin X alone
and treated with mitochondrial toxin X and rescues compound Y. The
fluorescence values obtained measuring TMRM were corrected for the
cell number.
Figure 4 shows that the assay can reliably be scaled up to a 384 well
plate format and gain robust, reproducible results.
Fig. 4: Fluorescence intensity values indicating the membrane potential in
control fibroblast in 384 well plate (192 wells used). The values were
obtained either after treatment with mitochontrial toxin X alone or
mitochondrial toxin X and rescue compound Y.
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